Validation of a method for the detection and confirmation of nitroimidazoles and the corresponding hydroxy metabolites in pig plasma by high performance liquid chromatography–tandem mass spectrometry

Nitroimidazoles (Ronidazole, Dimetridazole, Metronidazole, Ipronidazole) and their hydroxy metabolites are banned substances with antibiotic and anticoccidial activity. They are suspected to be carcinogenic and mutagenic. Since nitroimidazoles showed an inhomogeneous distribution and a rapid degradation in incurred muscle samples, plasma is the preferred target matrix for residue analysis. The analytical method of Polzer et al. [J. Polzer, C. Stachel, P. Gowik, Anal. Chim. Acta 521 (2004) 189] was adapted for liquid chromatography–tandem mass spectrometry detection and was validated in house according to the Commission Decision 2002/657/EC. The method is specific for all nitroimidazole except for Ipronidazole and its metabolite, due to interferences at their retention times in chromatograms of blank plasma and reagents samples. The absence of a matrix effect enables the use of a (linear) calibration curve in solution for quantitation. The apparent recovery (obtained after correction with a deuterated internal standard) is between 93% and 123%, except for the metabolite of Metronidazole (58–63%). The repeatability (CVr = 2.49–13.39%) and intralaboratory reproducibility (CVRW = 2.49–16.38%) satisfy the Horwitz equation. The obtained values for the detection capacity (CCβ) range from 0.25 to 1 μg L−1, while values obtained for the decision limit (CCα) are below CCβ.